Our goals are to identify and characterize the susceptibility genes for autoimmune thyroid diseases (AITD), Graves' disease (GD) and Hashimoto's thyroiditis (HT), and to dissect the genetic-epigenetic causes of thyroid autoimmunity. In the last grant cycle, we made substantial progress toward these goals: (1) We fine mapped loci linked with AITD and identified 2 new AITD genes: ARID5B and NRXN3; we also identified new genes associated with distinct subsets of AITD; (2) We discovered that arginine at position 74 of the DRb1chain is key to susceptibility to AITD; (3) We found that a GD-associated TSHR variant in intron 1 down regulated TSHR transcription by epigenetic interaction with the transcription factor PLZF; (4) We identified a thyroglobulin (Tg) promoter variant that predisposes to AITD, and showed that the risk allele interacted epigenetically with IRF-1 to modify histone signatures; (5) We defined CD40 as a major GD susceptibility gene and demonstrated that the susceptible allele increased thyroidal expression of CD40 triggering GD via activation of IL-6. Following these discoveries our hypothesis is that susceptibility genes trigger AITD through alterations in gene expression and/or function as a result of genetic-epigenetic interactions. Our specific aims are: Specific Aim 1: To identify rare genetic variants causing AITD by performing next generation sequencing (NGS) in our unique dataset of 102 multiplex AITD families that are enriched with such variants. We will perform NGS on 2 previously replicated linked loci (10q and 14q) in members of linked families. We will use structural modeling to identify variants predicted to alter gene function/expression; these selected variants will be tested in a large cohort of AITD patients and controls. Specific Aim 2: To dissect genetic-epigenetic interactions of AITD genes with interferon alpha (IFNa). We will test the hypothesis that genetic-epigenetic interactions between a thyroglobulin (Tg) promoter SNP and IFNa trigger AITD by accelerating Tg synthesis and inducing ER stress. We will also test if a TSHR intron 1 SNP predisposes to GD by facilitating binding of PLZF to intron 1 thereby decreasing thymic TSHR synthesis, leading to escape from central tolerance. Specific Aim 3: We will test the hypothesis that a CD40 Kozak SNP predisposes to GD by upregulating CD40 expression in the thyroid when there is local thyroidal inflammation. We will also dissect the CD40 signaling pathways in thyroid cells, and translate these findings into new therapies for murine GD using CD40 blockade. In summary, our project builds directly on the knowledge gained in the last grant period, and focuses on 3 AITD genes that we and others identified, Tg, TSHR, and CD40. We now propose to move from gene-discovery to functional analyses and to dissect the mechanisms by which these genes trigger AITD. Our multidisciplinary team has the experience and expertise to achieve our aims. Our functional studies will unravel the molecular mechanisms causing AITD. It is the understanding of these mechanisms that will lead to translational research testing novel, mechanism-based, treatment and prevention strategies such as cytokine and CD40 blockade.